Safety and efficacy in geese of a PER.C6-based inactivated West Nile virus vaccine
Introduction
West Nile virus (WNV) was first identified in 1937 in the West Nile district of Uganda [1], and the first human outbreaks were reported in 1950 in Israel and 1974 in South Africa. These first outbreaks were associated only with mild flu-like symptoms with mortality rates close to zero [2], [3], [4], and as a result WNV was never considered as a serious threat to human populations. In 1999, a deadly variant of the WNV emerged in New York and spread rapidly into and across North America [5]. During this period, WNV has infected more than 120,000 individuals in the US leading to many hospitalizations. Since an effective human vaccine against WNV is not yet available, massive aerial spraying programs with pesticides aimed at eradicating the mosquito vectors were executed with limited effect. This aggressive emergence of WNV has prompted research laboratories to develop effective candidate vaccines for animal populations, notably horses, susceptible wild and domesticated avian species, and humans. The human disease caused by WNV (strain NY99) is characterized by fever, nausea and headache, and in many instances is further accompanied by diarrhea. In severe disease, WNV infects motor neurons in the brainstem resulting in the loss of neuron function. This leads to severe encephalitis and meningitis with mortality rates of 5–13%, rising to 15–30% in the geriatric population, children, and the immunocompromised [6], [7], [8], [9]. In horses, WNV causes a neurological disorder involving the spinal cord alone or the entire nervous system [10], [11]. Among the domesticated avian species affected by WNV, the goose is highly suscepible to natural infection. Clinical signs include ataxia, drooped wings and paresis. Recumbent geese are unable to stand and will die from secondary infection. In some young flocks up to 60% become affected and die [12]. Here, we show that WNV strains can be efficiently propagated on the mammalian PER.C6 cell line [13], and after inactivation and formulation with an adjuvant, the WNV vaccine demonstrates excellent safety in geese. Moreover, we show that vaccinated geese are fully protected against an otherwise lethal WNV challenge. Finally, we demonstrate here that protection of the geese correlates with antibody levels induced by the inactivated vaccine. These studies provide a scientific basis for the further development of an inactivated WNV vaccine based on strain NY99 which can then be tested in equines and in human clinical trials.
Section snippets
Virus strains and phylogenetic relationships
WNV strain ISR98 has been described previously [14] and was isolated in 1997 and 1998 from a dead goose in Israel [15]. Strains NY99 (snowy owl 385-99), AUS60 (MRM16 KUN), AUS91 (K6453 KUN), CYP68 (Q3574-5), and MAD78 (DakAnMg798) were provided by Dr. Robert Shope (University of Texas Medical Branch, Galveston, USA). To verify strain origin PCR primers were designed flanking the prM/E (nucleotides 549–1828) and NS5 (nucleotides 7681–10,395) genes as identified from GenBank entry AF196835
WNV replication in PER.C6® cells and estimation of goose and mouse LD50
To investigate whether WNV can replicate on mammalian PER.C6 cells we assessed the optimal inoculation dose of ISR98 on the cell line and showed that one WNV infectious particle per 104 PER.C6 cells causes 100% cell kill within 3 days (Fig. 1A). We subsequently determined the peak titers of six different WNV strains on PER.C6 cells. Although all tested strains could be propagated, the highest titers (mean log10 titer of 9.7 TCID50/ml) were attained for the neuro-invasive strains NY99 and ISR98 (
Discussion
The data obtained here demonstrate that the PER.C6 cell line provides an excellent platform for the production of an inactivated WNV vaccine. The major advantages of using PER.C6 cells include the excellent documentation of the origins of the cell line and its safety record which facilitates regulatory acceptance of products derived from this cell line. Also, it has been shown that this cell line can be scaled to grow in very large volumes, at high cell densities, and in diverse cell culture
Acknowledgements
The authors thank Dr. Robert Shope (University of Texas Medical Branch, Galveston, USA) for sending strains NY99, AUS60, AUS91, CYP68 (Q3574-5), and MAD78. The authors also thank Guiseppe Marzio and Marco Oerlemans (Crucell Holland BV) for technical support. This work was in part funded by SenterNovem grant IS04210.
References (30)
- et al.
A neurotropic virus isolated from the blood of a native of Uganda
Am J Hyg
(1940) - et al.
West Nile fever—the clinical features of the disease and the isolation of West Nile virus from the blood of 9 human cases
Am J Hyg
(1954) The ecology of West Nile virus in South Africa and the occurrence of outbreaks in humans
Ann NY Acad Sci
(2001)- et al.
West Nile in the Mediterranean basin: 1950–2000
Ann NY Sci
(2001) - et al.
West Nile virus: epidemiology and clinical features of an emerging epidemic in the United States
Annu Rev Med
(2006) Vector surveillance for West Nile virus
Ann NY Acad Sci
(2001)West Nile encephalitis in Russia 1999–2001: were we ready? Are we ready?
Ann NY Acad Sci
(2001)- et al.
Clinical characteristics of the West Nile fever outbreak, Israel, 2000
Emerging Infect Dis
(2001) - et al.
West Nile fever outbreak, Israel, 2000: epidemiologic aspects
Emerg Infect Dis
(2001) - et al.
West Nile encephalitis
Vet Clin North Am Equine Pract
(2000)
Clinical signs of West Nile virus encephalomyelitis in horses during the outbreak in Israel in 2000
Vet Rec
An inactivated West Nile virus vaccine for domestic geese-efficacy study and a summary of 4 years of field application
Vaccine
New helper cells and matched early region 1-deleted adenovirus vectors prevent generation of replication-competent adenoviruses
Hum Gene Ther
Origin of the West Nile virus responsible for an outbreak of encephalitis in the northeastern United States
Science
Virus encephalomyelitis of geese: some properties of the viral isolate
Isr J Vet Med
Cited by (27)
Efficacy assessment of an inactivated Tembusu virus vaccine candidate in ducks
2017, Research in Veterinary ScienceProtection of red-legged partridges (Alectoris rufa) against West Nile virus (WNV) infection after immunization with WNV recombinant envelope protein E (rE)
2013, VaccineCitation Excerpt :Similar results have also been described in geese, as 3–10 weeks-old birds were susceptible to the infection, while older animals were not [35]. In agreement with previous reports [27,28], no specific IgG were detected in any animal after a single immunization, but all rE vaccinated partridges were ELISA positive after two immunizations. In contrast, as expected, none of the sham-immunized birds had specific antibodies.
Domestic goose as a model for West Nile virus vaccine efficacy
2013, VaccineCitation Excerpt :An inactivated WNV vaccine produced from suckling mouse brain inactivated with formaldehyde and blended with mineral oil adjuvant produced 86% protection from mortality in laboratory experimental studies and 75% protection in farm studies after intracerebral challenge in geese [18]. However, this vaccine protection study and several others used an intracranial challenge [12,18,19], instead of subcutaneous used in the current study; i.e. the current study better mimics the natural route of infection by the mosquito bite and may be better predictive of vaccine protection in the field. For subcutaneous WNV challenge in a goose experimental model, potential metrics for measuring vaccine protection (Table 5) could include preventing or reducing clinical disease, detecting vaccine induced antibodies to the virus, detecting a decrease in challenge virus within the plasma, and reducing histopathological changes and viral antigen deposition in brain and heart which have been reported as pathological features in previous pathogenesis studies [8].
Susceptibility of the PER.C6 cell line for infection with clinical human respiratory syncytial virus isolates
2012, Journal of Virological MethodsAttenuated West Nile viruses bearing 3′SL and envelope gene substitution mutations
2008, VaccineCitation Excerpt :Efforts to develop human and veterinary vaccines against WNV have taken both traditional and novel approaches. Formalin-inactivated whole virus vaccines have been approved for use in horses [12] and tested successfully in geese [13]. DNA vaccines encoding the WNV structural proteins have also been assessed for veterinary use and have been found to be protective in mice, horses and birds [14,15].